During the past year we have made considerable progress in identifying downstream effectors of the Raf-1 signalling pathway. We have identified the first Raf-1 substrate, mitogen-activated protein-kinase-kinase (MAP- KK). We have recapitulated the Raf-induced kinase cascade in vitro using purified components (i.e., activated Raf-1 overexpressed from baculovirus vectors in SF9 cells phosphorylated and activated purified MAP-KK which then phosphorylated and activated p44-MAP kinase). This activated MAP kinase efficiently phosphorylated c-Jun in vitro at residues adjacent to the DNA binding domain and phosphorylated c-Myc at residues within the amino terminal 100 amino acids. In addition, we have uncovered an action of Raf-1 that appears to be independent of MAP kinase activation. In Jurkat T-cells Raf induced interleukin-2 production without concomitant extracellular signal-regulated kinase activation. Lastly, we have shown that ultraviolet light (UV) and Raf-1 induce the phosphorylation (at serines 63 and 73) and activation of c-Jun in vivo. A dominant-negative Raf mutant blocked Ras- and Src-induced phosphorylation of c-Jun at these sites, indicating a requirement for Raf-1 in this signalling pathway. The dominant-negative Raf mutant also blocked Src-, Ras-, UV-, phorbol ester- and serum-induced expression of a reporter gene driven by Ap-1/Ets or SRE binding sites. Furthermore, dominant-negative MAP kinase and Jun mutants blocked activated Raf-induced transformation of NIH3T3 cells. Thus, our results indicate that Raf-1 functions in a linear signal transduction pathway with the order Src-Ras-Raf-1-MAP-KK-MAP kinase-c-Jun/Ets.